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Valentina KALICHUK Valentina KALICHUK Mémoire présenté en vue de l’obtention du Grade de Docteur de l'Université de Nantes et de l’Université Catholique de Louvain Écoles doctorales: Biologie-Sante (UN) et Secteur des sciences de la santé (UCL) Discipline: Biomolécules, pharmacologie, thérapeutique (UN) et Sciences biomédicales et pharmaceutiques (UCL) Spécialité: Biologie des organismes (UN) et Sciences pharmaceutiques (UCL) Unité de recherche : Inserm U1232 – CNRS – 6299 – CRCINA et Louvain Drug Research Institute IRS-UN, Team 13: Nuclear Oncoloy Research Advanced Drug Delivery and Biomaterials Soutenue le 16/10/2017 A novel generation of Affitins for targeting cancer cells with drug-loaded lipid nanocapsules JURY Président du Jury : Nick DEVOOGDT, Professeur des universités, ICMI, Vrije Universiteit Brussel, Belgium Rapporteurs : Nick DEVOOGDT, Professeur des universités, ICMI, Vrije Universiteit Brussel, Belgium Vladimir TOLMACHEV, Professeur des universités, IGP, Uppsala University, Sweden Examinateurs : Diego ARANGO, Directeur de Recherche,Vall d’Hebron Hospital Research Institute, Barcelona, Spain Olivier FERON, Professeur des universités, FATH, Université Catholique de Louvain, Belgium Pierre SONVEAUX, Professeur des universités, FATH, Université Catholique de Louvain, Belgium Directeurs de Thèse : Frédéric PECORARI, Chargé de Recherche, Inserm U1232, Université de Nantes, France Véronique PREAT, Professeur des universités, LDRI, Université Catholique de Louvain, Belgium A novel generation of Affitins for targeting cancer cells with drug-loaded lipid nanocapsules Valentina V. Kalichuk Supervisors: Dr Frédéric Pecorari and Prof Véronique Préat With the support of: Папе. (To my father.) Table of contents Acknowledgements 3 Foreword 5 Abbreviations 7 Chapter I: Introduction 11 1. Cancer and the selectivity challenge in medicine 15 2. Molecular targeting in cancer 19 3. 7 kDa DNA-binding proteins as alternative scaffolds 45 4. The Epithelial cell adhesion molecule as a target in cancer therapy 59 5. Nanoparticles in cancer therapy 69 Chapter II: Aims of the thesis 107 Chapter III: The archaeal “7 kDa DNA-binding” proteins: extended characterization of an old gifted family 113 Chapter IV: A novel, smaller scaffold for Affitins: Showcase with binders specific for EpCAM 151 Chapter V: Affitin-functionalized lipid nanocapsules for targeting colorectal cancer cells 187 Chapter VI: Conclusion and perspectives 229 Scientific communications 241 Curriculum vitae 243 1 Acknowledgements The accomplishment of this thesis would not have been possible without the support of my family, friends and my professional and personal mentors. Foremost, I want to express my sincere gratitude to my supervisors Dr. Frédéric Pecorari and Prof. Véronique Préat for believing in my skills and giving me the opportunity to work under their guidance. They have been not only open to share their knowledge and discuss ideas, but also always supportive and patient with me. I would like to thank the members of my thesis committee Prof. Olivier Feron and Dr. Patrick Chames for the insightful discussions and encouragement. I am also grateful to the members of my jury Prof. Vladimir Tolmachev, Prof. Nick Devoogdt, Prof. Pierre Sonveaux and Prof. Diego Arango for agreeing to evaluate this work. During the last years I had the opportunity to work with colleagues from three different labs. First I want to thank my group in France: Stanimir, Petar, Benjamin, Axelle, Barbara, Georgi and Dessi for their friendship and support. I am especially grateful to Ghislaine, who was always there to help and to share her enormous experience and chocolate mousse. Furthermore I thank the other members of Team 13 that have supported me – Michel, Maxime, Sébastien, Stefanie, and others. I would also like to thank all the people who made my stay in Nantes a nice experience: Swapnil, Neha, Johann, Magali, Edouard, Benoit, Iyanar, Marine, Simon, 2T, Nadege, Kevin B., Kasia, Maxime J., Rémi, Celine, Kubat etc. Special thanks go also to the Teze family – Françoise, Hubert, Florence, Marie, Jeremie and Sam. I will always be especially grateful to David, who supported me through each step of my life and work during the last years. 3 Moving to Brussels, I was lucky to work with people like Bernard, Kevin, Nathalie and Murielle, who are always there to help with a smile, no matter how busy they are. I want to express my gratitude to Fabienne for her supervision, support and trust. I thank Chiara, Dario, Alessandra, Nikos and Thibaut not only for their help in the lab, but also for their support as friends. Same goes for Janske, Hanane, Carmen, Cécile, Audrey, Neha, Ana, Natalija, Sohaib, Mengnan and all the others, with whom we shared so many beautiful (and delicious!) moments together. I am grateful to Yoann, who did not let me give up even for a second and always had my back. I want express my sincere gratitude towards John-Ivan, who was, and still is, my guardian angel. He and Gergana became like a family to me. I would also use the opportunity to thank Prof. Andreas Plückthun, who accepted me for an internship in his lab. He has motivated me with his expertise and never-ending pool of ideas. I want to thank Sheena, Hännschen, Jonas, Brandy, Wolfgang, Dominik, David V. and the others who showed me how working hard and having fun should always go hand in hand. Special thanks to Kevin and Caramel-the-cat for chasing away the demons while I was writing and for always finding a way to put the smile back on my face. No words can express my gratitude towards my family and friends back home and around the world. Their constant support, unconditional love and care help me every day to do my best. Every progress and every small success in my life belongs to them. 4 Foreword A progressive strategy against cancer is the targeting of tumour- associated antigens by specific ligands coupled to nanoparticles, carrying therapeutic or imaging agents. Antibodies are the most widely used targeting molecules, but they possess limitations as high production costs, complex structure and limited stability. Affitins are highly stable engineered affinity proteins, derived originally from Sac7d, an archaeal polypeptide from the 7 kDa DNA-binding family (also known as Sul7d family). These binders show comparable affinity and specificity to those of antibodies, while being thermally and chemically more stable, cheaper to produce, easier to engineer and present a simpler structure and 20-fold smaller size. Lipid nanocapsules (LNCs), prepared by solvent free process, possess great stability and high efficiency for lipophilic drugs encapsulation and protect the drug from rapid degradation. Targeting LNC to cancer cells can further decrease drug concentration in normal tissues and lower the toxicity. The aim of the thesis is to combine the advantages of Affitins as targeting agents and LNCs as carriers in order to create vehicles for delivering payloads to cancer cells. The first goal of this work is to characterize more extensively the Sul7d family and to identify a potential candidate for the generation of Affitins with improved properties. The second goal is to validate the chosen affinity scaffold by creating binders against EpCAM and to characterize them. The last goal is to attach the new binders as affinity moieties to LNCs and to assess the tumour targeting of these complexes in vitro. 5 6 Abbreviations Abbreviations Ab Antibody ABD Albumin binding domain ADC Antibody-drug-conjugates ADCC Antibody-dependant cellular toxicity Ag Antigen AR Ankyrin repeat BSA Bovine serum albumin CD Circular dichroism CDC Complement-dependant cytotoxicity cDNA Complementary DNA CDR Complementarity-determining region CEA Canceroembryogenic antigen CH Constant domain of a heavy chain of an antibody CL Constant domain of a heavy chain of an antibody CTC Circulating tumour cells CSC Cancer stem cells ct-DNA Calf thymus DNA DARPins Designed Ankyrin Repeat Proteins DiD 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine perchlorate DNA Deoxyribonucleic acid DOX Doxorubicin 7 Abbreviations DSC Differential scanning calorimetry DSPE-PEG 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [amino(polyethylene glycol) EDTA Ethylene-diamine-tetraacetic acid EGFR Epidermal growth factor receptor ELISA Enzyme-linked immunosorbent assay EMA European Medicines Agency EMSA Electrophoretic mobility shift assay EMT Epithelial-to-mesenchymal transition EpCAM Epithelial cell adhesion molecule EpEX Extracellular domain of EpCAM EpICD Intracellular domain of EpCAM EPR Enhanced permeability and retention Fab/Fv Variable, antigen-binding fragment of an antibody FACS Fluorescence-activated cell sorting FDA Food and Drug Administration GFP Gren fluorescent protein HCAb Heavy-chain antibodies HER2 Human epidermal growth factor receptor 2 His6 Hexahistidine tag hrEpCAM human recombinant EpCAM HRP Horseradish peroxidase HSA Human serum albumin HWEL Hen white egg lysozyme IgG Immunoglobulin G IMAC Immobilized-metal ion affinity chromatography 8 Abbreviations IPTG Isopropyl β-D-1-thiogalactopyranoside KD Dissociation konstant kDa Kilodalton LC-ESI-HRMS Liquid chromatography-electrospray ionization-high resolution mass spectrometry LNC Lipid nanocalsules mAbs Monoclonal antibodies MNPs Magnetic nanoparticles MRI Magnetic resonance imaging mRNA Messenger ribonucleic acid MSA Mouse serum albumin MSNs Mesoporous silica nanoparticles Nb Nanobody NK Natural killer (cells) NP Nanoparticle PAGE Polyacrylamide gel electrophoresis PBS Phosphate-buffered saline PCR Polymerase chain
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